Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
  • C08H1/00—Macromolecular products derived from proteins
  • C08H1/06—Macromolecular products derived from proteins derived from horn, hoofs, hair, skin or leather
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24

Definitions

• the present invention relates to a biocompatible functional material, a method for producing the same, a medical device, a crosslinking agent, a surgical treatment method, and a regenerated tissue.
• One of the treatment methods for patients whose nerve tissue has been cut due to accidents, disasters or other reasons is to connect a tube made of artificial material to the nerve defect and induce nerve tissue regeneration in the tube. Have been done.
• a tube made of silicone, polyurethane, polyester, polyethylene terephthalate, alginic acid, polylactic acid, or the like, coated on its inner surface with a cell adhesive protein such as collagen-laminin is used.
• a cloth made of synthetic polymer fibers such as silicone, polyurethane, and polyester is made into a tube, and the inner surface is made of a cell-adhesive protein such as collagen-laminin.
• a method of transplanting an artificial blood vessel coated with the above into a blood vessel cutting site and inducing endothelial cells inside the artificial blood vessel has been performed.
• tubes and artificial blood vessels to be implanted in animals are required to have elasticity that is linked to the movement of the human body and each tissue.
• Young's modulus of tubes and artificial blood vessels made mainly of silicone or polyester is required.
• the Young's modulus of the adaptive tissue (elastic modulus) is 1 X 1 0 4 ⁇ 2 X 1 0 6 Pa Material whereas, having order at 1 x 1 0 7 P a or, Ri come caused a strong stress to the junction, so that is a problem, such as blood clots, the same elasticity as human tissue was required.
• the present inventor has conducted intensive studies in view of the above-mentioned problems of the prior art, and as a result, cross-linked water-soluble elastin with a cross-linking agent to remove cell-adhesive proteins such as collagen-laminin.
• the inventors have found that a crosslinked elastin having elasticity sufficient to be suitable for transplantation of a living body can be obtained without separation, and the present invention has been completed based on this finding.
• the present invention has the following configuration.
• a crosslinked elastin obtained by crosslinking a crosslinked raw material containing at least one selected from water-soluble elastin with a water-soluble crosslinking agent obtained by crosslinking a crosslinked raw material containing at least one selected from water-soluble elastin with a water-soluble crosslinking agent.
• the raw materials for crosslinking are collagen, gelatin, fibronectin, fibrin, laminin, casein, keratin, sericin, thrombin, proteins, polyglutamic acid, polyamino acids polylysine, polygalacturonic acid, heparin, Chondroitin sulfate, hyaluronic acid, dermatan sulfate, chondroitin, dextran sulfate, sulfated cellulose, alginic acid, dextran, carboxymethyl chitin, galactomannan, arabic gum, tragacanth gum, dielan gum, sulfated dielan, Karaya gum, carrageenan-agar, agar, xanthan gum, potato dolane, pullulan, cellulose, starch, carboxymethylcellulose, methylcellulose, soy-soluble polysaccharide, darcomannan, chitin, Chitosan, xyloglucan, lentinan carbohydrate,
• the average diameter of the void is 20 ⁇ !
• the elastin crosslinked product according to (5) which has a size of about 2 mm.
• a water-soluble cross-linking agent is a water-soluble compound having a hydrophobic portion in the molecular center region and having an active ester group that reacts with an amino group at both ends.
• R or R 3 is either ⁇ A> or ⁇ B> represented by the following structural formula, and and R 3 may be the same or different.
• R 4 and R 5 are any of H, CH 3 and C 2 H 5 , and R 4 and R 5 may be the same or different.
• R 3 is either ⁇ A> or ⁇ B> represented by the following structural formula, and and R 3 may be the same or different, and
• R 4 and R 5 are any of H, CH 3 and C 2 H 5 , and R 4 and R 5 may be the same or different.
• FIG. 1 is an electron micrograph of the crosslinked elastin of the present invention of Example 8 (reacted at 25 ° C.).
• FIG. 2 is an electron micrograph of the crosslinked elastin product of the present invention of Example 9 (reacted at 50 ° C.).
• FIG. 3 is a diagram showing the elastin 1% gelatin cross-linked molded article of Example 10 ⁇
• FIG. 4 is a view showing a crosslinked molded article of elastin 10% gelatin of Example 11.
• FIG. 5 is a view showing a cross-linked molded article of elastin 90% gelatin of Example 12.
• FIG. 6 is a view showing a crosslinked molded article of elastin 0% / gelatin of Example 13.
• FIG. 7 is a comparison photograph of elastin 0 to 90% / cross-linked gelatin.
• FIG. 8 is a view showing a crosslinked molded article of elastin containing heparin of Example 14.
• FIG. 9 is a diagram showing a test for confirming heparin content in Example 15;
• FIG. 10 is a diagram showing a sheet-like crosslinked elastin product of Example 16.
• FIG. 11 is a view showing a crosslinked sheet-like elastin of Example 17.
• FIG. 12 is a diagram showing a crosslinked fibrous elastin product of Example 17.
• Fig. 13 shows the pelleted elastin cross-linked product of Example 17.
• c Fig. 14 shows the growth curve of neuroblastoma cells (IMR-32) on cell adhesion proteins.
• FIG. The symbols are gelatin (m), elastin ( ⁇ ), albumin (mouth), No is the initial cell number on the protein-coated culture plate, and Nt is the cell number at the time of measurement.
• FIG. 15 is a diagram showing a fibroblast growth factor-containing elastin / heparin cross-linked product of Example 19.
• the water-soluble elastin used in the present invention is not particularly limited, but is obtained by hydrolyzing elastin, and specifically, is obtained by treating an animal's cervical ligament with heat oxalic acid.
• ⁇ -elastin or /? — elastin obtained by treating elastin with alkaline ethanol at least elastin, water-soluble elastin enzymatically treated with elastinase, and at least tropoelastin, a precursor in the elastin biosynthesis pathway
• One or more elastins can be used.
• the tropoelastin is not particularly limited, and at least one tropoelastin gene product obtained by a genetic recombination method can be used in an extract from animal cells.
• Elastin which is an elastic protein, is usually abundant in the body, such as the aorta and vocal cords, in the body where elasticity is required.
• Exist in the living body Elastin has water-insoluble properties due to its high content of hydrophobic amino acids and strong cross-linking structures such as desmosine and isodesmosine.
• the elastin has elasticity due to the formation of a unique structure called an oily coil due to such a crosslinked structure.
• the crosslinked elastin of the present invention can be obtained by cross-linking at least one selected from water-soluble elastin which has been made water-soluble by decomposing the crosslinked structure of elastin in vivo with a water-soluble crosslinker.
• the elastin molded article of the present invention can be obtained by mixing the above-mentioned water-soluble elastin and a water-soluble cross-linking agent to form a water-soluble elastin aqueous solution, then pouring the mixture into a molding template or the like, and cross-linking by heating.
• the crosslinked elastin of the present invention may contain a third component other than water-soluble elastin and a crosslinking agent.
• the components are not particularly limited. Examples of the components include proteins such as collagen, gelatin, fibronectin, fibrin, laminin, casein, keratin, sericin, and thrombin; polyamino acids such as polyglutamic acid and polylysine; polygalacturonic acids; and heparin.
• extracellular matrix components such as collagen, gelatin, fibronectin, laminin, heparin, and chondroitin sulfate, and cell growth factors such as bFGF (basic fibroblast growth factor) are preferable for enhancing cell adhesion and proliferation.
• bFGF basic fibroblast growth factor
• the proportion of water-soluble elastin contained in the crosslinked elastin of the present invention is preferably in the range of 0.5 to 99.5% by weight based on the crosslinked elastin. It is more preferably 1 to 95%, and within this range, a molded article having elasticity suitable for a living body and good moldability can be obtained.
• Water-soluble elastin is a hydrophobic protein composed of about 94% of the total weight by hydrophobic amino acids and about 1% by amino acids containing amino groups in the side chain (lysine, arginine, histidine).
• the water-soluble cross-linking agent used in the present invention may be any water-soluble cross-linking agent as long as it reacts with the amino group on the side chain of water-soluble elastin and undergoes a cross-linking reaction.
• Examples of the water-soluble crosslinking agent include glutaraldehyde, ethylene glycidyl ether, and the like, and compounds having a hydrophobic portion in the molecular center region represented by the following general formula and having an active ester group at both ends. be able to.
• a compound represented by the following general formula is used as a crosslinking agent, a molded article having elasticity suitable for a living body and good moldability is preferably obtained.
• R or R 3 is either ⁇ A> or ⁇ B> represented by the following structural formula, and R j ⁇ and R 3 may be the same or different.
• ⁇ O (ri is an integer from 1 to 20.)
• the water-soluble cross-linking agent of the present invention has a dicarboxylic acid compound represented by the above general formula, wherein both terminals are activated with 4-hydroxyphenyldimethyl-sulfoniummethylsulfate (4-hydroxyphenyldimethyl-sulfoniummethylsulfate: DSP). It has elastin, which contains a large amount of hydrophobic amino acids, and a hydrophobic portion which has a strong and stable structure, and has the characteristic that it can be dissolved in water and handled in an aqueous system.
• the active ester groups at both ends of the chemical formula are peptide-bonded to the amino acid of water-soluble elastin and cross-linked. Therefore, the crosslinked elastin obtained by crosslinking with the water-soluble crosslinking agent of the present invention has a characteristic that it is susceptible to biodegradation in a living body. Since the biodegradation rate is related to the degree of cross-linking of the crosslinked elastin, it can be controlled by changing the cross-linking conditions and the degree of cross-linking.
• the structure of the crosslinked elastin of the present invention is not particularly limited, but is preferably a sponge structure having voids so that a body fluid, a culture solution, or the like can penetrate.
• the size of the void is not particularly limited, but when the average diameter is less than 20 zm, a hard crosslinked body having a high Young's modulus (elastic modulus) is easily obtained.
• the thickness is in the range of 20 to 2 mm, a moldable crosslinked body having a low Young's modulus (elastic modulus) and a high degree of swelling can be easily obtained.
• Crosslinked elastin of the present invention is a crosslinked product excellent in elasticity, Young's modulus in order to facilitate a biocompatible preferably in the range of (modulus) lxl 0 2 ⁇ lxl 0 7 P a, in particular 1 X 1 0 range of 3 ⁇ 2 X 1 0 6 P a is preferable.
• the shape of the elastin molded article of the present invention is not particularly limited, but is preferably in the form of a thread, a film, a rod, a pellet, a tube, or the like for application to medical use.
• the crosslinked elastin of the present invention alone forms a specific structure. Or a complex with components other than the crosslinked elastin. Further, it may be used for surface coating of a structure other than the elastin crosslinked body.
• the components that form the complex are not particularly limited, but include, for example, proteins such as collagen, gelatin, fibronectin, fibrin, laminin, casein, keratin, sericin, thrombin, polyglutamic acid, and polylysine.
• the conditions for the cross-linking reaction between the water-soluble elastin and the water-soluble cross-linking agent are not particularly limited, but the reaction temperature should be in the range of 4 to 150 ° C under normal pressure or under pressure such as in a autoclave. Is preferred. In particular, the range of 10 to 120 ° C. is preferable from the viewpoint of operability of crosslinking.
• the crosslinked elastin of the present invention In the case of a sponge having voids, the diameter of the voids can be controlled by controlling the reaction temperature.
• reaction temperature when the reaction temperature is in the range of 4 to 50 ° C, a crosslinked body having an average pore diameter of 20 m or more can be obtained, and in the range of 50 to 150 ° C, the average pore diameter is 2 mm.
• a crosslinked product of less than 0 zm can be obtained.
• the method for molding the crosslinked elastin of the present invention is not particularly limited, but it can be obtained by using a molding die used for molding a general synthetic resin.
• a water-soluble elastin and a water-soluble cross-linking agent of the present invention are mixed to form a water-soluble elastin aqueous solution, then poured into a molding machine, and heated and cross-linked with an automatic crepe.
• Elastin cross-linked products such as membranes, rods, pellets or tubes can be obtained
• the crosslinked elastin of the present invention has excellent elasticity because it has elasticity in the same region as the living body, and can be effectively used as cosmetics and medical devices.
• cosmetics it can be used as a base material for a face mask as a skin care product.
• a medical device a device formed and applied to a component such as a catheter, a shunt, a wound dressing, etc., provides an unprecedented flexible function ( and the use of these materials as a regenerative medical material).
• the medical device of the present invention is implanted in the body, the target tissue easily grows smoothly in the body, and is particularly effective for the nerve cells and blood vessels described above.
• an elastin crosslinked product and a third component can be added to impart functions that are not inherent to elastin.
• heparin II cell growth factor which is antithrombotic and interacts with cell growth factor, can be included.
• third components may be added as a raw material in advance to form a crosslinked body when preparing an elastin crosslinked body, or after forming the elastin crosslinked body, impregnating the structure, or Dry and physically It may be adsorbed. Still further, the third component may be chemically immobilized on the crosslinked elastin in order to suppress delamination.
• the crosslinked elastin of the present invention can also be used as a sustained-release carrier, which is one of DDS (Drag Delivery System).
• the crosslinked elastin product of the present invention can provide a sponge structure having a high Young's modulus (elastic modulus) and a void, and can exhibit an effect in treating nerves, blood vessels, and the like.
• the medical device of the present invention has the functions and effects as described above, it is particularly effective when used for medical treatment.
• a collagen sheet having a blood coagulation component such as fibrin or thrombin fixed to one side of a sheet is used as an adhesive for hemostasis after surgery.
• the crosslinked elastin of the present invention can be embedded in the body as described above and used as a place for regenerating blood vessels and nerves, but this function can also be used outside the body. That is, as a culture substrate for regenerative medicine, transplanting and culturing oriented embryonic stem (ES) cells, somatic stem cells, mesenchymal stem cells, etc., on the membrane surface or tube of the present invention. Thus, it is possible to form an organ of a desired form. Since the crosslinked elastin of the present invention is not only good in moldability but also biodegradable, it is cultured to a certain extent, and a cartridge-type regenerative medical method and regeneration for transplanting an organ that has formed a shape together with a culture substrate Provide organization.
• the hydroxyl group of dicarboxylic acid was activated esterified with 4-hydroxyphenyldimethyl-sulfoniummethylsulfate (4-hydroxyphenyldimethyl-sulfoniummethylsulfate: DSP).
• DSP Active esterification by the method described in peptide chemistry (K. Kouge, T. Koizumi, H. Okai, and T. Kato. (1987) Bull. Chem. Soc. Jpn., 60, 2409.
• reaction solution (filtrate) was added dropwise to a 70 mL (70 ml) solid to solidify, and the solid was dried under reduced pressure to obtain 1.4 g of the water-soluble crosslinking agent [A] of the present invention.
• the purity of the obtained crosslinking agent was 98% as measured by 1H-NMR (JNM-EX-500, JE0L).
• FIG. 1 shows a cross section of the obtained elastin molded article taken with a scanning microscope at a magnification of 90 times. From the electron micrograph, the internal structure of the crosslinked elastin was a sponge structure having voids, and its average diameter was 62 m. Void Table 2 shows the modulus of elasticity of the crosslinked elastin of different types.
• Experimental Example 9 (Elastin molded article was prepared using the water-soluble cross-linking agent [A] obtained in Experimental Example 7 as the cross-linking agent and changing the cross-linking conditions)
• FIG. 2 shows a photograph of a section of the obtained elastin molded article taken with a scanning microscope at a magnification of 90 times. From the electron micrograph, the internal structure of the elastin crosslinked product was a sponge structure with voids, and its average diameter was 9 m.
• Deionized water 148 1 was added to 72 mg of the water-soluble elastin and 8 mg of gelatin obtained in Experimental Example 1 to dissolve, and then the water-soluble cross-linking agent 278 mM N 39 l prepared in Experimental Example 7 (the cross-linking agent was elastin amino group content).
• the cross-linking agent was elastin amino group content.
• a 30% aqueous solution of elastin was prepared. The aqueous solution was poured into a mold and heated with an autoclave at 120 ° C. for 30 minutes to obtain a crosslinked elastin and its molded product (FIG. 5).
• the swelling is large when the elastin content is 0% (swelled in water to about 2.3 times the diameter of the template after 6 hours), but contained 1%
• a well-formed gel was formed from the gel (Fig. 7: 0,1,10,90% containing elastin).
• the swellability of the other gels was about 1.5 times that of the template with 1% gel, about 1.4 times that of the template with 10% gel, and about 1.1 times that of the template with 90% gel.
• the gel thus prepared was thoroughly washed with deionized water, and then stained in a 1% toluidine- “0” aqueous solution. Toluidine blue "0” stains blue-violet when bound to heparin. From FIG. 9, it was confirmed that heparin was incorporated into the gel.
• a mold for molding was prepared using two pieces of slide glass treated with lipelsilane (silicon coated) and a silicone rubber sheet as a spacer.
• the 30% water-soluble elastin aqueous solution obtained in Experimental Example 1 was used.
• a solution containing the water-soluble cross-linking agent (3 times the molar amount of elastin amino groups) prepared in Experimental Example 7 was poured in to prevent air and water from entering from outside. While maintaining this state, the substrate was heated at 80 ° C. for 30 minutes in water to obtain an elastin film (FIG. 10).
• Experimental Example 17 (Preparation of various molded products of elastin)
• An elastin membrane (thickness 0.5 mm, lcmx lcm) was placed on a plastic plate (6 holes) for tissue culture, 2 ml of the culture solution was added, and the mixture was allowed to stand at 37 ° C for 30 minutes.
• the culture solution was dissolved by adding 215 ml of deionized water to 2.57 g of MEM Hanks powder and adding 1.17 ml of baking soda solution (7.5%), 2.5 ml of glutamine solution (200 mM), and 2.5 ml of non-essential amino acid solution. 5 ml and 25 ml of fetal calf serum were added.
• neuroblastoma cells Neuroblastoma, IMR-32 (ATCC No. CC-127)
• concentration of 1.0 ⁇ 10 4 cel / ml was performed and the cells were incubated at 37 ° C. for 24 hours. After the incubation, the number of cells was counted over time using a cell counting plate or direct observation.
• Experimental Example 1 9 (Elasta containing fibroblast growth factor (FGF) and heparin Water soluble elastin 75 mg and heparin 5 mg were dissolved in deionized water 148, and then dissolved in Experimental Example 7. Add 39 ⁇ l of cross-linking agent (278 mM) to make a 30% aqueous solution of elastin, pour the solution into a mold and heat at 120 ° C (autoclave) for 30 minutes to react.
• FGF fibroblast growth factor
• the crosslinked elastin of the present invention is a material having elasticity suitable for living body transplantation without delamination of the cell-adhesive protein, and thus can be used as a conventional material. It has the effect of solving the problem of detachment of the cell-adhesive protein during the long-term treatment that has occurred and the problem of insufficient regeneration of tissues such as nerves and blood vessels.
• the water-soluble cross-linking agent of the present invention cross-links water-soluble elastin, and if there is a ⁇ type, a cross-linked elastic elastin that can be formed in any shape can be obtained. It is processed into pellet, tube, and even regenerative medicine materials and medical device materials, and has the effect of being able to be used in a wide range of applications.
• the crosslinked elastin of the present invention forms a sponge structure having voids, it can easily penetrate drugs and the like, and can be easily combined with other materials, and provide a new medical material. It has the effect of making it possible.

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• 2002
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